The Section on Connective Tissue Disorders studies the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which primary collagen defects cause skeletal fragility and other significant connective tissue symptoms and then to apply the knowledge gained from these studies to the treatment of children with these conditions. The Section conducts an integrated program of laboratory and clinical investigation. Children with types III and IV OI form a longitudinal study group. In turn, skin and bone samples from our patients provide material for mutation identification and investigations of collagen processing, fibrillogenesis and osteoblast biology. This arrangement allows us to integrate data in a manner that is unique among OI research programs. We have generated a knock-in murine model for OI that carries a classical OI mutation in type I collagen. We have named this mouse the Brittle (Brtl) mouse. A point mutation causing a gly349cys substitution was introduced into one murine col1a1 allele. This substitution was modelled on the collagen defect present in one of our type IV OI patients. We have been engaged in investigations of the skeletal adaptation of the Brtl mouse during puberty, using a combination of physical modalities, including bone densitometry, biomechanics and histomorphometry. It is well-known that fractures in type IV OI decrease after puberty but the mechanism of this change is unknown. Bone density (BMD) of the Brtl femur and spine is about 70% of wild type prior to puberty. After puberty, the BMD of the wild type mice does not change significantly. However, the post-pubertal Brtl mouse does have a significantly greater BMD than the pre-pubertal Brtl, attaining 90% of wild type BMD. Thus, a change in the amount, composition or organization of the mineral phase of the skeleton occurs during puberty in the Brtl mouse; this change is distinct from pubertal development in the wild type mouse. Biomechanical studies also suggest that the increased strength of the Brtl bone in the context of weak geometry points to changes in the composition of the Brtl bone material itself as the key to post-pubertal adaptation. Understanding the physiological processes that cause improved bone strength and the mechanisms that control them may provide novel approaches to the therapy of OI. The Brtl mouse has also been used for studies examining the effect of the bisphosphonate alendronate on OI bone. This is a collaboration with investigators at HSS who are treating a recessive mouse model for OI, the oim mouse, using an identical protocol to allow direct comparison of oim and Brtl data. We have taken a mutation suppression approach to the gene therapy of the dominant negative connective tissue disorders. Suppression of the level of mutant collagen transcripts would, in principle, transform a structural collagen mutation with severe clinical consequences into a quantitative mutation with mild to undetectable clinical symptoms. We are using hammerhead ribozymes as the mutation suppression agent. We have previously demonstrated the specificity and effectiveness of hammerhead ribozymes in vitro and in cultured OI cells. We are currently engaged in generating transgenic mice with the ribozyme under tet control to achieve high levels of ribozyme expression. These mice will be bred to the Brtl mice for investigation of the effect of ribozyme in collagen mutations and the OI phenotype in vivo. Parents who are mosaics for the collagen mutations that cause clinically significant OI in their children are models for the cell therapy approach to OI. Although these individuals carry the collagen mutation in a fraction of their cells, they are clinically normal or minimally affected. In cell therapy, a portion of the osteocytes in an affected individual would be replaced with normal cells, resulting in a mosaic individual who might have improved skeletal function. One major deficit in the scientific rationale for cell therapy has been a lack of information about the proportion of heterozygous bone cells present in genetic mosaic individuals. We have examined skeletal cells from two asymptomatic mosaic carriers with COL1A1 mutations. Each mosaic carrier has a high proportion of dermal fibroblasts that are heterozygous for the collagen defect that causes OI in their children. A carrier for type IV OI had the collagen mutation in 75% of osteoblasts cultured from a site on her left side and 50% of osteoblasts cultured from a site on her right side. A carrier for type III OI had the collagen mutation in 40% of calvarial cells and about 65% mutant cells in such type I collagen rich tissues as lung, trachea and aorta. Both women had normal bone histology and minimal clinical signs. Thus, our data provide the first demonstration that a significant burden of mutant osteoblasts is compatible with normal bone growth, density and histology, allowing us to tentatively set the goal for cell therapy of OI at 50-60% normal cells. We have been investigating the consequences of a rare type of collagen mutation on collagen assembly, stability and incorporation into fibrils and matrix. We delineated a triplet duplication in COL1A1 exon 44; the normal allele encodes three identical Gly-Ala-Hyp triplets, while the mutant allele encodes four. This mutation shifts the register of the collagen chains with respect to each other but does not interrupt the triplet sequence and yet it causes a lethal phenotype. The realignment of X and Y positions caused by the register shift delays helix formation, causing overmodification. The register shift persists throughout the entire helix and decreases the rate of N-proteinase processing. The register shift also disrupts incorporation of mutant collagen into fibrils and matrix. Collagen helices with two mutant chains and a significant portion of helices with one mutant alpha1(I) chain do not participate in fibril formation. This exclusion of mutant chains would be expected to cause dramatically decreased matrix production in vivo. In matrix deposited by proband fibroblasts in culture, mutant chains were well-incorporated into the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. We have been conducting a four-arm treatment trial of the bisphosphonate pamidronate and recombinant growth hormone (rGH). Children with types III and IV OI are randomized among four groups - pamidronate alone, rGH alone, both drugs or no drugs. Since growth hormone stimulates osteoblasts to produce bone matrix and the bisphosphonate inhibits resorption by osteoclasts, the two drugs could act synergistically to produce increased quantities of bone matrix. This NICHD study is the only controlled trial of pamidronate in OI children. Major endpoints include lumbar spine bone density and vertebral compressions. Because it is possible that increased quantities of bone matrix containing abnormal type I collagen might lead to increased brittleness of bone, we will particularly focus on delineating whether the quality of the bone matrix is improved. This study is currently in progress.